Notes: The authors characterized a novel caspase 8-like activity that cleaves Bid and activates the mitochondrial apoptotic pathway. This activity was purified from rat liver lysosomal extracts and later identified as chymotrypsin B (CtrB). CtrB was previously thought to be expressed only in the pancreas, but the authors were able to detect crtB RNA in total RNA from primary rat hepatocytes and a rat hepatoma cell line (RH-35) using RT-PCR and the Access RT-PCR System. To confirm the intralysosomal localization of Crt B, the authors transfected RH-35 cells with an expression vector encoding CrtB tagged with green fluorescent protein. Prior to transfection, synthesis of functional protein from the expression vector was confirmed by in vitro transcription and translation using the TNT^® Coupled Transcription Translation System and [³⁵S] methionine. (3889)

Notes: These authors studied the effect of specific amino acid substitutions in the N^pro gene of swine fever virus. The N^pro gene encodes a non-strucutral protein that prevents interferon production by promoting proteasomal degradation of interferon regulatory factor 3 (IRF3). N^pro also has an autoprotease function. Deletion of the entire N^pro region attenuates virulence. In this study, the authors showed that degradation of IRF3 and autoprotease activity are independent, structurally overlapping functions. In particular, they investigated the effect of specific amino acid substitutions that eliminated IRF3 interaction and degradation, but did not affect autoprotease activity. They showed that removal of IRF3 degradation activity of N^pro had only minimal effect on virulence in swine. The pGEM-T Vector was used to clone the amplified N^pro gene, and the CheckMate™ Flexi Vector Mammalian Two-Hybrid System was used for protein interaction studies. (3944)

Notes: The authors characterized masculinizing mutations of the female-promoting tra genes in Caenorhabditis briggsae (Cb-tra). Using RT-PCR, the authors monitored the levels of full-length Cb-tra mRNA and a novel splice variant; actin mRNA was amplified as a control. RT-PCR was carried out using the AccessQuick™ RT-PCR System and RNA from 5–10 worms per 50µl reaction. (3892)

Notes: HEK-293 were transiently transfected with adenovirus 5 DNA using FuGENE® 6 Reagent. Cells were seeded at 3 × 10⁶ cells/75 cm² and grown to 60% cell confluency prior to transfection. Cell transfections were 60–70% efficient. (4266)

Notes: The authors of this paper compared time-resolved, fluorescence energy transfer and ATP-based luminescent assays in ultrahigh-throughput screens (1536-well) for Rho-associated kinase II inhibitors. They found that both technologies are suitable for such a screen and perform similarly; however, they note that the ATP-based luminescent kinase assay provides an economical advantage. (3932)

Notes: The authors sought to detect expression of the chemokine CXCL16 and its receptor, CXCR6, in prostate cancer cell lines and in benign and malignant prostate cancer tissues. The Access RT-PCR System was used to amplify and detect CXCL16 and CXCR6 mRNA in these cells and tissues. Each RT-PCR contained 1µg of total RNA, and amplifications were carried out for 35 cycles. Amplified products were detected on a 1.5% ethidium bromide-stained agarose gel. (3836)

Notes: HepG2 cells in a T-25 culture flask (3 million cells at 70ndash;80% confluency) were transfected with CYP3A4 reporter plasmid (a pGL3 reporter construct) and a CMV-Renilla control plasmid (a total of 2.5µg of plasmid mix was used) using FuGENE® 6 Reagent for transient transfection assays. Reporter activities were measured using the Dual-Glo® Luciferase Assay System. (4268)

Notes: To examine the role of cyclin-dependent kinase (cdk)-3 expression in cancer cell lines, potential targets of cdk3 phosphorylation were examined. The CheckMate™ Mammalian Two-Hybrid System was used to test for transcription factor binding partners of cdk in HEK293 cells. Cell proliferation of T96G cells stably transfected with either cdk3 or vector was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3983)

Notes: The authors identified the maize homolog of hcf136 (Zmhcf136), a gene involved in photosynthesis, and used an RNA blot to determine if ZmHcf136 transcripts accumulate preferentially in mesophyll cells. DNA probes for Zmhcf136 and several cell-specific markers were generated by PCR using GoTaq^® Green Master Mix, gel purified and radiolabeled prior to use in the RNA blots. To examine differences in protein accumulation and localization in wildtype and hcf136 mutants, proteins from subcellular fractions were subjected to two-dimensional gel electrophoresis, and spots of interest were excised, digested with Sequencing Grade Modified Trypsin, then analyzed by electrospray ionization-tandem mass spectrometry. (3883)

Notes: Somatic cell nuclear transfer technique was used to generate human blastocyst-stage embryos using nuclei from adult male fibroblasts cell lines and enucleated oocytes. Genomic DNA was analyzed using the PowerPlex® 16 system to confirm the genetic identity of the blastocyst cells. (3952)

Notes: The authors describe the developmental validation of the Plexor^® HY System, a quantitative PCR assay that simultaneously quantifies total human and male DNA. Validation studies examined: (1) human specificity, (2) sensitivity, (3) quantitation of degraded DNA, (4) impact of inhibitors, (5) male/female mixture and Y-assay male specificity, (6) reproducibility and concordance and (7) population studies. (3969)

Notes: Regulation of genes targeted by the ligand-activated aryl hydrocarbon receptor (AhR) has been shown to be controlled by calcium (Ca(2+)) changes induced by AhR agonists such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This study investigated this link. As part of the study, MCF-7 cells were transfected with various pGL3 firefly luciferase reporter constructs and the control pRL-TK Vector expressing Renilla luciferase. Transfection conditions were as follows: MCF-7 cells were cultured in 24-well plates and transfection was performed using FuGENE® 6 transfection reagentwith a FuGENE:DNA ration of 3:1. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. (4362)

Notes: The authors of this study created a cDNA library representative of the extracellular proteome (secreted proteins and the extracellular domains of transmembrane proteins). Each cDNA was individually transfected into 293T cells. Medium from the cDNA of each transfection was used in a suite of cell-based assays. The CellTiter-Glo^® Assay was used to screen for secreted factors from the cell lines expressing the cDNA that affected viability of twelve cell lines: human primary B cells, human primary T cells, human primary NK cells, human primary monocytes, A549 cells, Colo205 cells, U-118 cells, MDA-MB231 cells, PC3 cells, PANC1 cells, human primary skeletal muscle progenitor cells, and rat primary oligodendrocyte precursor cells. (3935)

Notes: The authors performed protein pull-down assays to characterize the interaction of ORAI1 and STIM1, two protein components of the calcium-release calcium current. His₆-STIM1 C terminus and ORAI1 were synthesized using the TNT® Coupled Reticulocyte Lysate System in the presence of ³⁵S, and His₆-STIM1 C terminus was immobilized using MagZ™ Binding Particles. An aliquot of the TNT® reaction expressing ORAI1 was added to the particles, and proteins were washed, eluted using increasing concentrations of imidazole (10–40mM) and analyzed by SDS-PAGE. In a second set of pull-down assays, His₆-STIM1 C terminus was used to pull down ORA1 N- and C-terminal fragments expressed as GST fusion proteins. The His₆-STIM1 C terminus protein was purified from transiently transfected HEK293 cells using the MagneHis™ Protein Purification System. (3781)

Notes: One potential source of antibiotic-resistant bacteria is food-producing animals. The authors examined the ability of protegrin-1 (PG-1), an antimicrobial peptide, to protect wildtype and transgenic mice expressing PG-1 against bacterial infection. As part of the cloning strategy to produce the PG-1 expression construct, the authors amplified and cloned full-length PG-1 into the pGEM^®-T Easy Vector. To test the bactericidal activity of PG-1 expressed in transgenic mice, radial diffusion assays were performed, in which test samples were added to a well containing E. coli and the clear antibacterial zone was measured. Two of the test samples were neutrophil secretions from the PG-1 transgenic mice and purified polyhistidine-tagged PG-1 protein, purified using the MagneHis™ Protein Purification System. (3896)

Notes: Genomic DNA from Arabidopsis thaliana was isolated using the Wizard® Genomic DNA Purification Kit. The extracted DNA was used to amplify the 3´ UTR of AteIF5A-2, A. thaliana translation initiation factor 5A, or the entire gene for creating transgenic plants. Leaf discs from wild-type Arabidopsis were infected with Pseudomonas syringae pv tomato DC3000, a virulent strain regulated by AteIF5A-2, using a syringe. After 24 and 72 hours, 0.4cm leaf discs were fixed, labeled using the DeadEnd™ Fluorometric TUNEL System and stained for AteIF5A-2 protein. (3979)

Notes: These authors used the Flexi^® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag^® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi^® System and Gateway^® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag^® fusion proteins was consistent with results obtained using GFP-fusions. (3800)

Notes: The authors designed a high-throughput gene-expression screen to identify compounds that induce a neuroblastoma gene signature in BE(2)-C cells. The screen used Valproic Acid (VPA), an inhibitor of histone deacetylase, as an enhancer. The top hit from the screen was all-trans retinoic acid (ATRA). The authors proposed that ATRA + VPA would reduce cell viability and might induce apoptosis by inducing genes associated with terminal differentiation. They used the CellTiter-Glo^® Luminescent Cell Viability Assay in a 96-well format to assess the effects of ATRA and a variety of inhibitors of histone deacetylase on BE(2)-C cell viability. (3931)

Notes: Lynch syndrome (LS) is due to mutations in at least four DNA mismatch repair (MMR) genes. There are two tumor characteristics that can be screened for in an attempt to identify patients with CRC who are most likely to have LS. These characteristics are microsatellite instability (MSI) and loss of one or two of the MMR proteins in the tumor compared to normal tissue. MSI testing and IHC for the four mismatch repair proteins was performed on 500 tumors from unselected patients with CRC. If either MSI or IHC was abnormal, complete mutation analysis for the mismatch repair genes was performed. GoTaq Master Mix (Cat.# M7122, Cat.# M7132) (4111)

Notes: The authors of this study describe a reporter gene system that allows researchers to create one genetic construct that can be used for a variety of studies including imaging and protein immobilization. The HaloTag® reporter protein is engineered to form covalent bonds with ligands that have different functional reporters. (3925)

Notes: The authors examined the heritability of primary angle closure using a classic twin study with both monozygotic and dizygotic twins. Zygosity of same-sex twins was determined by typing 15 STR loci using the PowerPlex^® 16 System. (4042)

Notes: The authors examined the heritability of certain eye characteristics that may be associated with glaucoma using a classic twin study with both monozygotic and dizygotic twins. Zygosity of same-sex twins was determined by typing 15 STR loci using the PowerPlex^® 16 System. (4043)

Notes: The authors examined strains of Salmonella enterica serovar Typhi isolated from typhoid cases originating in or around Indonesia or from travelers returning from Indonesia to examine if serovar Typhi from this area has a greater level of genetic diversity compared to other countries. Genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit, diluted to 4 ng/µl and used in locus-specific PCR genotyping. (3980)

Notes: Thees authors searched for small-molecule inhibitors of galactokinase (GALK). They developed an HTS assay using the Kinase-Glo^® Assay System. The HTS assay used 15 μM ATP and α-D-galactose as the substrate and was performed in 384-well plates against 50,000 small molecules. Two hundred compounds were identified from the primary screen as GALK inhibitors. (3934)

Notes: The authors describe an HTS assay to screen for inhibitors of measles virus infection. The CytoTox 96^® Non-Radioactive Cytotoxicity Assay was used to assess toxicity of all confirmed hit compounds from the primary screen. A luminescent assay was used to assess cell-to-cell fusion in the presence candidate compounds that appeared to inhibit entry. Effector cells that expressed the T7 polymerase and measles virus H and F envelope proteins were overlaid on target cells expressing firefly luciferase under the control of a T7 polymerase promoter. Inhibition of fusion should reduce luciferase expression compared to a positive fusion control. Ten of the eleven compounds tested caused a dose-dependent reduction in luciferase expression, suggesting they block viral entry into cells. Luminescence was detected using the Bright-Glo™ Assay System. (3933)